Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix.

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Measurement of plasma and platelet tissue factor pathway inhibitor, factor V and Protein S in people with haemophilia.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B. AIM: To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding. METHODS: Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors. RESULTS: There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S. CONCLUSION: Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.

Translation of human tissue factor pathway inhibitor-ß mRNA is controlled by alternative splicing within the 5' untranslated region.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3' splice variants, TFPIα and TFPIß, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5' untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated. APPROACH AND RESULTS: Exon 2 splicing occurs in TFPIα and TFPIß transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPIß translation but not on TFPIα. Luciferase reporter assays further exposed strong translational repression of TFPIß (90%) but not TFPIα. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPIß. Exon 2 also repressed luciferase production (80% to 90%) when paired with the ß-actin 3' untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPIα 3' untranslated region. CONCLUSIONS: Exon 2 is a molecular switch that prevents translation of TFPIß. This is the first demonstration of a 5' untranslated region alternative splicing event that alters translation of isoforms produced via independent 3' splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue-specific regulation of TFPIß-mediated anticoagulant activity.

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi-/- mice die during embryonic development. The absence of protease-activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi-/-mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. OBJECTIVES: To determine biological functions of pltTFPIα. METHODS: Tfpi-/-/Par4-/- mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. RESULTS: Recipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin-positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. CONCLUSIONS: Tfpi-/-/Par4-/- mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Factor V east Texas variant causes bleeding in a three-generation family.

BACKGROUND: The factor V east Texas bleeding disorder (FVETBD) is caused by increased plasma tissue factor pathway inhibitor-α (TFPIα) concentration. The underlying cause is a variant in F5 causing alternative splicing within exon 13 and producing FV-short, which tightly binds the C-terminus of TFPIα, prolonging its circulatory half-life. OBJECTIVES: To diagnose a family presenting with variable bleeding and laboratory phenotypes. PATIENTS/METHODS: Samples were obtained from 17 family members for F5 exon 13 sequencing. Plasma/platelet TFPI and platelet FV were measured by ELISA and/or western blot. Plasma thrombin generation potential was evaluated using calibrated automated thrombography. RESULTS: The FVET variant was identified in all family members with bleeding symptoms and associated with elevated plasma TFPIα (4.5- to 13.4-fold) and total TFPI (2- to 3-fold). However, TFPIα and FV-short were not elevated in platelets. TF-initiated thrombin generation in patient plasma was diminished but was restored by a monoclonal anti-TFPI antibody or factor VIIa. TFPIα localized within vascular extracellular matrix in an oral lesion biopsy from an affected family member. CONCLUSIONS: Factor V east Texas bleeding disorder was diagnosed in an extended family. The variant was autosomal dominant and highly penetrant. Elevated plasma TFPIα, rather than platelet TFPIα, was likely the primary cause of bleeding. Plasma FV-short did not deplete TFPIα from extracellular matrix. In vitro thrombin generation was restored with an anti-TFPI antibody or factor VIIa suggesting effective therapies may be available. Increased awareness of, and testing for, bleeding disorders associated with F5 exon 13 variants and elevated plasma TFPI are needed.

Intrauterine lethality in Tfpi gene disrupted mice is differentially suppressed during mid- and late-gestation by platelet TFPIα overexpression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein required for murine embryonic development. Intrauterine lethality of Tfpi-/- mice occurs at mid- and late gestation, the latter of which is associated with severe cerebrovascular defects. Megakaryocytes produce only the TFPIα isoform, which is stored within platelets and released upon activation. OBJECTIVES: To examine biological activities of platelet TFPIα (pTFPIα) by characterizing effects of pTFPIα overexpression in Tfpi-/- mice. METHODS: Transgenic mice overexpressing pTFPIα were generated and crossed onto the Tfpi-/- background. Genetic and histological analyses of embryos were performed to investigate the function of pTFPIα during embryogenesis. RESULTS: The transgene (Tg) increased pTFPIα 4- to 5-fold without altering plasma TFPI in adult Tfpi+/+ and Tfpi+/- mice but did not rescue Tfpi-/- mice to wean. Analyses of the impact of pTFPIα overexpression on Tfpi-/- survival, however, were complicated by linkage between the Tg integration site and the endogenous Tfpi locus on chromosome 2. Strain-specific genetic interactions also modulated Tfpi-/- embryonic survival. After accounting for these underlying genetic factors, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos but had no effect on development of cerebrovascular defects during late gestation resulting in their lack of survival to wean. CONCLUSIONS: pTFPIα overexpression rescued Tfpi-/- embryos from mid-gestational but not late gestational lethality. The prevalence of underlying genetic factors complicating analyses within our study illustrates the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results.

The contribution of TFPIα to the hemostatic response to injury in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Correlates of plasma and platelet tissue factor pathway inhibitor, factor V, and Protein S.

BACKGROUND: Plasma Tissue Factor Pathway Inhibitor (TFPI) circulates bound to factor V (fV) and Protein S (PS). Estrogen therapy decreases plasma TFPI and PS. TFPI, fV, and PS circulate within platelets, and are released upon activation to modulate thrombus formation. OBJECTIVE: Identify factors affecting the concentrations of plasma and platelet TFPI, fV, and PS. METHODS: Blood samples were obtained from 435 healthy individuals. Plasma total TFPI, TFPIɑ, fV, and PS, and platelet TFPI, fV, and PS were quantified. Correlations between these protein concentrations and age, gender, race, and estrogen use were established. RESULTS: In males, only plasma fV increased with age, while in females, all plasma analytes increased with age. Males had higher plasma total TFPI, TFPIα, and PS than females. The platelet proteins in either sex remained relatively stable with increasing age. Platelet TFPI and PS were comparable in both sexes, while platelet fV was higher in females. Estrogen use was associated with decreased plasma total TFPI and TFPIα, and platelet PS, but not with platelet TFPI concentration. Racial differences in plasma and platelet proteins were observed, some of which were larger than inter-individual differences observed within racial groups. TFPI, fV and PS concentrations correlated in plasma, while only fV and PS correlated in platelets. CONCLUSIONS: Plasma and platelet TFPI, fV and PS differ in their: (i) in vivo association; (ii) demographic correlates; and (iii) alteration by estrogen therapies. Therefore, the plasma and platelet pools of these proteins may modulate hemostasis and thrombosis via different biochemical pathways.

Plasma Proteolytic Cascade Activation during Neonatal Cardiopulmonary Bypass Surgery.

BACKGROUND: Neonates undergoing cardiopulmonary bypass (CPB) surgery to correct congenital heart defects often experience excessive bleeding. Exposure of blood to artificial materials during CPB may activate coagulation, complement and inflammatory pathways. In addition, the surgical stress placed on the haemostatic system may result in cross-activation of other plasma proteolytic cascades, which could further complicate physiological responses to the surgical procedure and post-operative recovery. Plasma protease inhibitors undergo distinct conformational changes upon interaction with proteases, and, thereby, can serve as endogenous biosensors to identify activation of the different proteolytic cascades. We tested the hypothesis that changes in the concentration and conformation of protease inhibitors regulating plasma proteolytic cascades during neonatal CPB are associated with post-operative bleeding. PATIENTS AND METHODS: Plasma samples from 44 neonates were obtained at four time points across the surgical procedure. Anti-thrombin, antitrypsin, anti-chymotrypsin, anti-plasmin, C1-inhibitor and tissue factor pathway inhibitor (TFPI) concentrations and conformations were evaluated by enzyme-linked immunosorbent assay, transverse urea gradient gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS/CONCLUSION: The most striking changes were observed following heparin administration and were associated with the appearance of inactive forms of anti-thrombin and an increase in the plasma concentration of TFPI. Changes in anti-thrombin and TFPI remained evident throughout surgery and into the post-operative period but were not different between patients with or without post-operative bleeding. The concentration of antitrypsin decreased across surgery, but there was no significant accumulation of inactive conformations of any inhibitor besides anti-thrombin, indicating that widespread cross-activation of other plasma proteolytic cascades by coagulation proteases did not occur.